Most industrial enzymes are currently produced by microorganisms via large-scale fermentation. Another approach to the production of such enzymes is to express and recover these enzymes from transgenic plants. Published work in this field has increased dramatically in recent years and several agricultural biotechnology companies have initiated research into using plants as “chemical factories” for the production of industrially useful proteins including pharmaceuticals. The appeal of this technology is the capability to produce large amounts of proteins as a value added product using conventional agricultural practices, and in an environmentally friendly manner. Indeed, high value proteins have been expressed in crop plants at commercially viable levels. Relatively low-value enzymes are more challenging to produce economically in plants but enzyme production costs can usually be offset somewhat by the lower degree of purity generally required.
Plastid transformation was first described in 1988, in the unicellular alga Chlamydomonas reinhardtii. Since then, plastids of various higher plants have been transformed, including tobacco, Arabidopsis, potato, rice, tomato, cotton, and carrot. Foreign DNA is generally introduced into plastids via microprojectile bombardment, although methods relying upon polyethylene glycol and microinjection have also been developed. The introduced DNA is flanked by regions of extensive homology to the integration site, facilitating recombination with the plastid genomic target site. In this respect, plastid transformation is fundamentally different from nuclear transformation methods, in which the transgene is inserted randomly into the nuclear genome. Another fundamental difference between the two transformation systems is the presence of many copies of the chloroplast genome (up to 10,000) per cell.
Transgenic chloroplasts offer a number of advantages over conventional transgenic plants. Among the primary advantages are high levels of transgene expression and foreign protein accumulation. Compared to nuclear transformation, where levels of recombinant protein expression in excess of 1% total soluble protein are relatively rare, plastid transformation frequently yields recombinant protein levels of 1-10%. This property has been exploited to produce high levels of heterologous proteins, e.g., human somatotropin in transplastomic tobacco plants (Staub J M et al., 2000, Nature Biotechnol 18: 333-338). Another advantage of this mode of plant transformation is that chloroplasts are generally not present in pollen (Medicago sp., Pinus sp., are exceptions to this rule), so that genetically engineered chloroplasts are less likely to spread into unmodified plants via cross-pollination.
Agro-based resources known as lignocellulosics are plant resources that contain cellulose, hemicelluloses, and lignin. Lignocellulosics include wood, agricultural residues, water plants, grasses, and other plant substances. Lignocellulosics such as agricultural and forestry wastes and crops produced specifically for biomass offer tremendous potential as a raw material for the production of fuel and chemical feed stocks.
Cellulose and hemicellulose are the principal sources of fermentable sugars in lignocellulosic feedstocks. A major challenge in utilization of this material is the conversion of polymeric cellulose to fermentable sugars. Acid hydrolysis is a relatively cheap process but yields of sugars are low. Enzymatic breakdown with cellulases (enzymes that break down cellulose to its simple sugar components) results in higher yields but is more costly, with enzyme production as the largest single component cost.
In an effort to lower the cost of cellulose production, it is desirable to produce cellulolytic enzymes in transgenic plants (U.S. Pat. Nos. 5,981,835; 6,818,803; U.S. patent application Pub. No. US2002/0062502 A1). Methods similar to these should further be explored. It would be beneficial to develop genetically engineered crop plants that produce economically viable levels of cellulases, and to develop the technology required to use these enzymes for biomass conversion.
The development of chloroplast transformation systems for crop plants and the high protein expression levels obtained with these systems suggests that chloroplast transformation may be a preferable way to achieve high expression levels of proteins. Such expression systems could make plant-based cellulase production economically viable. The invention described herein addresses this and other related needs.